HPLC - created from Mind Map

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ADVANTAGES

DISADVANTAGES

Selection of LC modes

HPLC HARDWARE

GENERAL STRUCTURE

DETECTORS

SENSITIVE

CAN BE AUTOMATED

QUANT ANALYSIS

GOOD AT SEPARATING VOLATILE/THERMALLY LABILE COMPOUNDS

WIDE RANGE OF SUBSTANCES: in/organic, metals, ions, proteins, aa,etc.

SEPARATE ON SIZE, CHARGE, POLARITY, SOLUBILITY

CHIRAL SEPARATIONS

+ WIDE RANGE OF DETECTORS

RECOVER SAMPLES

EXPENSIVE, IMPURITY FREE SOLVENT USED.

MOVING PARTS & CONNECTIONS

OPTIMAL PERFORMANCE = KEEP IT RUNNING REGULARLY

Mixing controller

Injector

Columns

Mix difft solvents in precise ratios

reqd for: ISOCRATIC= constant solvent composition. GRADIENT= changing solvent composition

mix diffnt solvents in precise ratios

make sure that solvents are miscible

loop & valve configuration

load & inject positions

flat ended syringe - manual injection

ensure the loop is entirely filled - inject sample in 1 plug (~ 3 rinses)

generally = stainless steel

Analytical column: ID- 1-5mm, particle size- 3-5um, length- 35-250mm, plates/m-40000-70000

typical: 4.6mm x 5um x 150m = 10000 plates/column

Microbore or 'rocket'

what's inside?

Monolithic (single crystal)

How do you choose?

short (30-75mm), thin (<4.6mm ID), small particles (3um) = faster

normal p.: polar stat, n-polar solvent (Si stst p.), 1:9 EtOAc:hexane mob p.

reverse p.: n-polar stat, polar solvent. (C18 stat p.), 15:85 MeOH:H2O mob p.

Polymer based, porous core, can control pore size

can run under lower pressure condition

better resolution, speed.

useful for larger mols.

partition Chrom.

adsorption chrom.

Ion Chrom

Size exclusion C. (gel permeation c.)

Affinity

Bonded Phases

OPTIMISING CONDITIONS (3 parameters)

stat. p. is a 2nd liquid immiscible w liq mob. p

nonionic, polar w low/moderate mol mass. (ionic cmpds w derivatization &ion pairing)

change mob. p. composition = enhance separation

SOLVENT STRENGTH

Normal Phase : k2/k1 = 10 ^(P'1-P'2)/2

if the ratio k2/k1 is large, need o have a big k2. if its small, - not resolved

k2: final retention factor

k1: initial "

Rev Phase: k2/k1 = 10 ^(P'2-P'1)/2

EFFECT OF MOB. P. ON α.

ION PAIR CHROM. (ANALYSTES THAT MAY EASILY IONISE) -org acids, amines.

CHIRAL SEPARATIONS

N : plate number

k : retention factor (strongly dependent upon make-up of mob. p.)

ideal range of k:2-10

complex mix: 0.5 - 20

α : can be changed by choosing diff column packing

α: selectivity factor

Strong solvent: interacts strongly w solutes (often polar)

Polarity index (P') - based upon solubility in 3 separate solvents, -a measure of relative polarity, -higher P' = stronger

Mixed solvent

P(AB)' = *AP'(A) + *BP'(B) *A= volume fraction of solvent A

manipulate chemi nature: - proton acceptor, proton donor, dipolar interactions

start to add small amt of acid or base.

in rev p. : aq solns of : THF, acetonitrile, methanol.

+ solvent modifier

2 xplanations

org salt w large org counter ion

added in small conc

counter ion pairs w analyte ion & this pair partition into stat. phase.

counter ion retained by the neutral stat. phase giving it a charge & get reversible ion-pair interactions w charged analyte

ceramic Mg silicate

cyclodextrin

Pirkle type chiral stat. phase. Works on π acceptor/ π donor interactions some H bonding.

separate chiral compounds - enantiomers

either mob/stat phase reqd to be chiral.

complexation btwn chiral resolving agent w one of the isomers results in separation.

active sites for cation exchange: sulfonate (strong acid) & carboxylic acid gp (weak)

Anionic exchangers - strongly basic tertiary amine gps / weak basic primary amine groups.

sulfonate based cationic resin equilibrated w NaOAc -injection of amines & alcohol

base materials: silica/zirconium/ organic polymer

with polymer columns: -know the pressure limitations of the packing, - packing may swell/contract in different solvents.

high mol mass sp.

packings (~10um) silica/polymer particles - a network of uniform pores

most stat. phases are crosslinked polymers

polymers may expand/contract in diffnt solvents.

Vtot = Vg + Vi + Vo

Vg= vol occupied by stat p.

Vi= vol of solvent held in pores

Vo= exclusion vol (vol solvent req to elute cmpnds too big for pores)

Vo = largest mol, Vi + Vo = smallest mols, Ve=Vo + KVi = intermediate mols (assume theres no mixing)

KVi : measure of th efraction of mols in the pores (K = Cs/Cm)

get finer separation by connecting columns in series. problems??

bond an affinity ligand to a neutral solid support

ligand reversibly and selectively bind to analyte (Ab, enz inhibitor)

mobile phase hv dual roles:

can wash out by changing ionic strength or changing pH

promote strong binding (analyte & affinity ligand)

release bound sp. when other cmpds hv been eluted

PHENYL ETHER (extremely polar aromatics)

SILICA

NITRO (aromatics & double bonds)

PHENYL HEXYL (aromatics)

SULFONIC ACID (weak bases)

QUATERNARY AMINE (nucleotides, nucleosides, organic acids)

DIOL (rev: gel filtration of peptides & proteins) (normal: = to silica, x deactivated by H2O)

CYANO (slightly polar)

AMINO (normal: alternate selectivity to silica) (rev: carbs) (ion xchange: anions & org'c acids)

IDEAL REQMNTS

3 basic types: optical, electrochemical, mass

1) REFRACTIVE INDEX

2) EVAPORATIVE LIGHT SCATTERING DETECTORS (ELSD)

3) FLUORESCENCE

4) UV & UV-VISIBLE (190-900nm)

5) PHOTODIODE ARRAY (PDA)

6) ELECTROCHEMICAL

7) MASS SPEC

adequate sensitivity

stable & reproducible

linear response over several orders of magnitude

short response time, independent of flow rate

similarity in response to all possible analytes

non-destructive

minimum internal volume

compatible w liquid flow

changes in DENSITY when analytes comesthrough cell

sensitive to turbulence, temp, solvent changes

used w isocratic systems only

fragile flow cells

effluent nebulised into fine mist -- through laser & scattered radiation detected at right angle to laser

volatile mob. phase.

evaportn tube is heated, but temp kept low - labile cmpds not degraded

sample excited by radiation from source (Hg, Xe, laser)

resulting fluorescence detected at 90° to excitn beam

can derivatise sample (add fluoro tag)

filter variable, fixed wavelength, wavelength variable

UV-VIS needs 2 lamps: a UV lamp - Hg, a visible lamp - tungsten

individual photosensitive elements : small silicon photodiodes (consist of a reversed-biased pn junction

4 electrochemical techniques: amperometry, voltammetry, coulometry, conductometry

Specific applications

respond to sp that can be red/ox

output is electron flow generated

3 electrodes : working, auxillary, reference

common ionisation sources : electrospray ionisation (ESI), Atmospheric Pressure Chemical Ionisation (APCI)